The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop

Peng, Xiao; Wang, Xiao; Wang, Hongmei; Xiaolei, Fu; Cui, Fuai; Yu, Xiao; Wen, Shishuai; Bi, Wenxiang; Sun, Junying

doi:10.1016/j.biocel.2014.10.004

Cited by 13 publications

(11 citation statements)

References 39 publications

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“…The interactions between PTPs and their substrate are mostly mediated by their catalytic domains. The binding of phosphosubstrates to the active sites of PTPs leads to the formation of the enzyme-substrate complexes (19). Domain-mapping experiments indicated that the N-terminal catalytic domain of PTPN1 and the cyclic dinucleotide binding domain (CBD) of MITA mediate their interaction (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

PTPN1/2-mediated dephosphorylation of MITA/STING promotes its 20S proteasomal degradation and attenuates innate antiviral response

Xia

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Upon cytosolic viral DNA stimulation, cGMP-AMP synthase (cGAS) catalyzes synthesis of 2′3′cGMP-AMP (cGAMP), which binds to the adaptor protein MITA (mediator of IRF3 activation, also called STING, stimulator of IFN genes) and induces innate antiviral response. How the activity of MITA/STING is regulated to avoid excessive innate immune response is not fully understood. Here we identified the tyrosine-protein phosphatase nonreceptor type (PTPN) 1 and 2 as MITA/STING-associated proteins. PTPN1 and PTPN2 are associated with MITA/STING following viral infection and dephosphorylate MITA/STING at Y245. Dephosphorylation of MITA/ STING leads to its degradation via the ubiquitin-independent 20S proteasomal pathway, which is dependent on the intrinsically disordered region (IDR) of MITA/STING. Deficiencies of PTPN1 and PTPN2 enhance viral DNA-induced transcription of downstream antiviral genes and innate antiviral response. Our findings reveal that PTPN1/2-mediated dephosphorylation of MITA/STING and its degradation by the 20S proteasomal pathway is an important regulatory mechanism of innate immune response to DNA virus.DNA virus | PTPN1 | PTPN2 | 20S proteasome | innate immune response

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Section: Resultsmentioning

confidence: 99%

PTPN1/2-mediated dephosphorylation of MITA/STING promotes its 20S proteasomal degradation and attenuates innate antiviral response

Xia

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Plasmids coding for His-tagged WT and mutant SSHs were transformed in E. coli BL21(DE3) cells and followed by isopropyl-1-thio-␤-D-galactopyranoside (IPTG) induction as described previously (46). When absorbance at 600 nm for cell cultures grown in lysogeny broth medium was between 0.6 and 0.8, IPTG was added to a final concentration of 0.1-0.3 mM, and the culture was further induced at 23°C for 12-16 h. The His 6 -tagged proteins were purified by Ni-NTA resin as described previously (44).…”

Section: Expression and Purification Of Ssh2mentioning

confidence: 99%

“…adjusted to 0.15 M by NaCl) as described previously (43,46), unless otherwise specified. All reactions were initiated by the addition of appropriate amount of SSH2 into the reaction mixtures containing different substrates at various concentrations, except that, in the experiment to examine the effect of F-actin on activation of SSH2 phosphatase activity, SSH2 (0.4 M) was preincubated with F-actin (4 M) at room temperature for 30 min before use.…”

Section: Allosteric Regulation Of Slingshotmentioning

confidence: 99%

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Allosteric modulation of the catalytic VYD loop in Slingshot by its N-terminal domain underlies both Slingshot auto-inhibition and activation

Du¹,

Peng

et al. 2018

Journal of Biological Chemistry

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Slingshots are phosphatases that modulate cytoskeleton dynamics, and their activities are tightly regulated in different physiological contexts. Recently, abnormally elevated Slingshot activity has been implicated in many human diseases, such as cancer, Alzheimer's disease, and vascular diseases. Therefore, Slingshot-specific inhibitors have therapeutic potential. However, an enzymological understanding of the catalytic mechanism of Slingshots and of their activation by actin is lacking. Here, we report that the N-terminal region of human Slingshot2 auto-inhibits its phosphatase activity in a noncompetitive manner. pH-dependent phosphatase assays and leaving-group dependence studies suggested that the N-terminal domain of Slingshot2 regulates the stability of the leaving group of the product during catalysis by modulating the general acid Asp in the catalytic VYD loop. F-actin binding relieved this auto-inhibition and restored the function of the general acid. Limited tryptic digestion and biophysical studies identified large conformational changes in Slingshot2 after the F-actin binding. The dissociation of N-terminal structural elements, including Leu, and the exposure of the loop between α-helix-2 and β-sheet-3 of the phosphatase domain served as the structural basis for Slingshot activation via F-actin binding and via neuregulin stimulation in cells. Moreover, we designed a FlAsH-BRET-based Slingshot2 biosensor whose readout was highly correlated with the phosphatase activities of Slingshot2. Our results reveal the auto-inhibitory mechanism and allosteric activation mechanisms of a human Slingshot phosphatase. They also contribute to the design of new strategies to study Slingshot regulation in various cellular contexts and to screen for new activators/inhibitors of Slingshot activity.

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“…In addition to the open and closed conformations of PTP1B, we also identify a semiclosed conformation (PDB entry 1QXK_A) which was previously clustered with the closed conformations in the analysis done by Tanchuk et al We also noted that in our PCA, ICA and NMF analyses, the PDB entry 2HNP_A (ligand‐free PTP1B) didn't cluster separately. In PCA and ICA, the PDB entry 2HNP_A clustered with a set of structures which had ligands bound (PDB entries 2HNQ_A, 3EAX_A, 3QKP_A, 4QAP_A, and 4QBW_A) The average RMSD of 2HNP_A to the other structures is 0.3 Å. 2HNQ_A has compound sodium tungstate at the active site, 3EAX_A has compound LZP‐6 at the active site, 3QKP_A has TRS and glycerol molecules at nonactive sites, 4QAP_A and 4QBW_A have TRS molecules at nonactive sites.…”

Section: Discussionmentioning

confidence: 99%

Structural analysis of protein tyrosine phosphatase 1B reveals potentially druggable allosteric binding sites

et al. 2018

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Catalytic proteins such as human protein tyrosine phosphatase 1B (PTP1B), with conserved and highly polar active sites, warrant the discovery of druggable nonactive sites, such as allosteric sites, and potentially, therapeutic small molecules that can bind to these sites. Catalyzing the dephosphorylation of numerous substrates, PTP1B is physiologically important in intracellular signal transduction pathways in diverse cell types and tissues. Aberrant PTP1B is associated with obesity, diabetes, cancers, and neurodegenerative disorders. Utilizing clustering methods (based on root mean square deviation, principal component analysis, nonnegative matrix factorization, and independent component analysis), we have examined multiple PTP1B structures. Using the resulting representative structures in different conformational states, we determined consensus clustroids and used them to identify both known and novel binding sites, some of which are potentially allosteric. We report several lead compounds that could potentially bind to the novel PTP1B binding sites and can be further optimized. Considering the possibility for drug repurposing, we discovered homologous binding sites in other proteins, with ligands that could potentially bind to the novel PTP1B binding sites.

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The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop

Cited by 13 publications

References 39 publications

PTPN1/2-mediated dephosphorylation of MITA/STING promotes its 20S proteasomal degradation and attenuates innate antiviral response

PTPN1/2-mediated dephosphorylation of MITA/STING promotes its 20S proteasomal degradation and attenuates innate antiviral response

Allosteric modulation of the catalytic VYD loop in Slingshot by its N-terminal domain underlies both Slingshot auto-inhibition and activation

Structural analysis of protein tyrosine phosphatase 1B reveals potentially druggable allosteric binding sites

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